Biomedical subjects
Publications and source records attributed to J F Hu.
Current anti-HIV drugs have extreme side effects and resistance to these drugs develops rapidly. The marine environment holds an unprecedented number of unusual chemical structural classes with activity against HIV. We review the literature on anti-HIV activity of marine natural products and discuss the efficacy of different structural classes.
Direct genetic manipulations have shown that telomerase-mediated telomere elongation plays a key role in determining cellular replicative capacity and senescence. The mechanisms regulating the production of an active telomerase enzyme are still predominantly unknown, although roles for transcriptional control of hTERT, alternative-splicing of hTERT transcripts, and post-translational phosphorylation of hTERT protein have been advocated. Here we show that hTERT is alternatively spliced in specific patterns by different tissue types during human development. Alternative splicing often prohibits the expression of hTERT protein containing functional reverse transcriptase domains. In these instances, telomerase activity is absent, leading to shortening of telomeres. The specific pattern of hTERT mRNA variants in human development provides evidence that alternative splicing is non-random and participates in the regulation of telomerase activity.
In this paper, the structure-activity relationships of chemically modified uronic acid polymer fragments from brown algae with regard to their antioxidant effects on H2O2-damaged lymphocyte were studied. The results indicated that the most potent antioxidant activity was obtained from the sulfated polysaccharide with ratio of mannuronate blocks (M-blocks) to guluronate blocks (G-blocks) of 3 to 1 and carboxyl residue unesterified. The sulfated G-blocks with esterified carboxyl residue also prevented lymphocyte from injury. However, the sulfated G-blocks bearing unesterified carboxyl residue hardly exerted antioxidant activity. These findings suggested that both M-blocks and esterified carboxyl residue were determinant structures in preventing lymphocyte from being oxidized by H202, indicating that the existence of M-blocks was more important in scavenging free radicals.
The acetylation of core histones can modulate the expression of numerous genes. In general, the deacetylation of histones results in transcriptional repression, whereas increases in histone acetylation lead to the enhancement of gene transcription. Since histone acetylation is maintained during mitosis, the acetylation pattern may contribute a heritable epigenetic imprint that can continue to influence gene transcription (1). These findings suggest that the degree of histone acetylation may represent one of several nonexclusive mechanisms that can initiate or maintain the allele-specific silencing of genomic imprinting. We have recently shown that the inhibition of histone deacetylation by Trichostatin A (TSA) induces the expression of the normally imprinted maternal IGF2 allele (2), leading to biallelic expression in both human and murine cells. Partial loss of imprinting of both sense and antisense Igf2r is also observed after TSA treatment. It has also been shown that exposure of mouse conceptuses to TSA leads to loss of H19 imprinting (3). In X-chromosome inactivation, the inactive chromosome is associated with relatively underacetylated histones (4). Changes in histone acetylation become apparent after the expression of Xist, the gene responsible for X-chromosome gene silencing, and after the downregulation of the inactivated X-chromosome genes (5), suggesting that decreased histone acetylation may mark or otherwise stabilize transcriptional repression rather than actually initiate it.
The mouse Gnas gene encodes an important signal transduction protein, the alpha subunit of the stimulatory G protein, G(s). In humans, partial deficiency of G(s)alpha, the alpha subunit of G(s), results in the hormone-resistance syndrome pseudohypoparathyroidism type 1a. The mouse Gnas (and the human GNAS1) locus is transcribed from three promoter regions. Transcripts from P1, which encode Nesp55, are derived from the maternal allele only. Transcripts from P2 encode Xlalphas and are derived only from the paternal allele, while transcripts from P3 encode the alpha subunit and are from both parental alleles. The close proximity of reciprocal imprinting suggests the presence of important putative imprinting elements in this region. In this report, we demonstrate that the reciprocal imprinting occurs in normal tissues of interspecific (Mus spretus x C57BL/6) mice. Transcripts from P1 are most abundant in CNS (pons and medulla) in contrast to the more ubiquitous expression from P2 and P3. In the P1-P2 genomic region, we have identified an antisense transcript that starts 2.2 kb upstream of the P2 exon and spans the P1 region. While the P1 transcript is derived from the maternal allele, the P1-antisense (Gnas-as) is derived only from the paternal allele in most but not all tissues. Although both the Nesp55 region and the Gnas-as transcripts are present in cerebral cortex, adrenal, and spleen, Gnas-as is abundant in some tissues in which transcription from the Nesp55 region is negligible. Furthermore, the Nesp55 region transcripts remain strictly imprinted in tissues that lack Gnas-as. Our results suggest that multiple imprinting elements, including the unique Gnas-as, regulate the allelic expression of the Nesp55 region sense transcript.
p73, a proposed tumor suppressor, shares significant amino acid sequence homology with p53. However, p73 is rarely mutated in tumors but it has been suggested that p73 is monoallelically expressed in some tissues. This latter feature would predispose p73 to gene inactivation because a single genetic 'hit' or the loss of the expressed parental allele would leave the cell without p73 activity. We examined the allelic expression of p73 in normal fetal tissues and in ovarian cancer and Wilms' tumor. We found that p73 was biallelically expressed in all fetal tissues, except in brain, where differential expression of the two parental alleles was observed. Biallelic expression of p73 was also observed in paired samples of ovary cancer and Wilms' tumor. Loss of heterozygosity of p73 occurred at relatively low rates in tumors: one of 11 informative samples (9.1%) of ovarian cancer and two of 19 (10.1%) Wilms' tumors. These data demonstrate that p73 is biallelically expressed in most tissues, thus excluding genomic imprinting as a molecular mechanism to predispose to allelic inactivation of p73 in human tumors.
The two contiguous IGF2 (human insulin-like growth factor II) and H19 genes are reciprocally imprinted in both human and mouse. In most tissues, IGF2 is transcribed only from the paternal chromosome while H19 is transcribed only from the maternal allele. The presence of a differential methylation region (DMR) on the two parental alleles at the 5' flanking region of H19 has been proposed to constitute the gametic imprint, which controls the reciprocal allelic expression of the two genes. Using bisulfite genomic sequencing, we have assessed the methylation status of cytosine (including 154 CpG sites) in six CpG-rich regions of the human IGF2-H19 genes. In a CpG island near promoter P3 of the IGF2 gene, more than 99.8% of all cytosines were converted to thymidine by sodium bisulfite mutagenesis, indicating that none of the CpGs was methylated. In the IGF2 exon 8-9 region, mosaic methylation of 56 CpG sites was observed in fetal tissues and in adult blood DNA. In contrast to the mosaic methylation of IGF2, the allelic methylation of the human H19 DMR was uniform. In the CpG region located 2 kb upstream (-2362 to -1911) of the H19 transcription site, all 25 CpG sites were completely methylated on only one parental allele. Uniform allele-specific methylation was also observed in the CpG island proximal to the H19 promoter (-711 to -290) with complete methylation of all 25 CpG sites in one parental allele. In contrast, the CpG region in the H19 promoter (-292 to +15) was mosaically methylated in all tissues. In addition, cytosine was methylated at three CpNpG and GpNpC sites on the top DNA strand and one CpNpG site on the bottom DNA strand from the fetal brain. The cytosines at CpG sites were methylated on both DNA strands (symmetric methylation) while cytosines at the CpNpG and GpNpC sites were methylated on only one DNA strand (asymmetric methylation). The asymmetric methylation was associated with tissue-specific disruption of H19 genomic imprinting in fetal brain.
Stimulus pattern related synaptic plasticity in the luminosity-type horizontal cell (LHC) of isolated carp retina was investigated. The major findings were: (1) repetitive red flashes progressively strengthened the synaptic connection between red-cone and LHC, whereas weakened that between green-cone and LHC; (2) repetitive green flashes remarkably depressed the LHC's red response, but caused little changes in the cell's green response. A competitive depression between different cone signals is suggested.
Telomerase extends telomeric repeats at the ends of linear chromosomes, thereby prolonging the replicative capacity of cells. To investigate possible regulatory mechanisms of telomerase, we measured telomerase enzyme activity, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in normal and neoplastic ovary, endometrium and myometrium. Telomerase activity was detected in most malignancies and in normal endometrium but not in myometrial leiomyoma, normal myometrium or normal ovary. hTR was expressed in all tissue samples. hTERT mRNA was expressed in many tissue samples, and no tissue sample exhibited telomerase activity without expressing hTERT mRNA. However, the presence of hTR and hTERT mRNA was not sufficient for telomerase activity. Alternate splicing of hTERT produced mRNAs lacking critical reverse transcriptase (RT) motifs in both normal and neoplastic tissues. Only tissues expressing hTERT containing complete A and B RT motifs demonstrated telomerase activity. Finally, several normal ovarian tissues and myometrial leiomyomas lacked telomerase activity despite expressing hTR and hTERT containing complete A and B RT motifs. This was not seen in ovarian and myometrial malignancies, where the expression of hTR and hTERT containing complete A and B RT motifs was sufficient for telomerase activity. We conclude that in ovarian and uterine tissues, the presence of a functional telomerase complex is regulated at multiple levels, including hTERT transcription and alternative splicing of hTERT transcripts. The lack of telomerase activity in several normal but not malignant tissues expressing hTR and hTERT containing complete A and B RT motifs suggests that there are further mechanisms for suppressing telomerase activity downstream of hTERT transcription and mRNA splicing, and these mechanisms have been lost during neoplastic transformation.
The mouse insulin-like growth factor II receptor (Igf2r) gene encodes two reciprocally imprinted RNA transcripts: paternally imprinted Igf2r sense and maternally imprinted Igf2r antisense. Although DNA methylation has been implicated in the initiation and maintenance of genomic imprinting, acetylation of core histones has recently been appreciated as another important factor that regulates gene expression. To determine whether histone acetylation participates in the regulation of Igf2r imprinting, we examined the relative abundance of acetylated histones in interspecific mice (M. spretus x C57BL/6). Oligonucleosomes derived from liver were immunoprecipitated with acetyl-histone antiserum and were analyzed for the allelic distribution of DNA from the region of the sense and antisense Igf2r promoters. In nucleosomes associated with the Igf2r sense promoter, histone acetylation was demonstrated on the maternal allele, which is transcriptionally active. There was much less histone acetylation on the suppressed paternal allele. In nucleosomes associated with the Igf2r antisense promoter, the active paternal allele was heavily acetylated, whereas the suppressed maternal allele was underacetylated. Treatment of cultured fibroblasts with the histone deacetylase inhibitor Trichostatin A induces partial relaxation of genomic imprinting as well as decreased DNA methylation of both Igf2r sense and antisense promoters. These results demonstrate that increases in histone acetylation can lead to decreased DNA methylation, thereby modulating the regulation of the imprinted expression of Igf2r sense and antisense transcripts.
Chemical screening with extracts of Streptomyces sp. (strain GT 61150) resulted in the detection, isolation, and structure elucidation of two new acyl alpha-L-rhamnopyranosides (1 and 2) and three new rhamnosyllactones A, B1 and B2 (3 approximately 5). Rhamnosyllactones B1 and B2 were obtained as a 5:1 mixture. The structures were confirmed by spectroscopic analysis, especially 2D-NMR techniques. The rhamnosyltransferase of our strain is able to connect the sugar moiety to heteroaromatic carboxylic acids and enols. The metabolites 1 and 4/5 as well as previously reported acylrhamnosides 6 approximately 11 inhibit the enzyme 3alpha-hydroxysteroid-dehydrogenase (3alpha-HSD).
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Two models have been proposed to account for the molecular mechanism underlying genomic imprinting of the insulin-like growth factor II receptor gene (Igf2r): expression-competition and promoter DNA methylation. To examine which model best explains the regulation of Igf2r imprinting, we examined the allelic expression of endogenous Igf2r sense and antisense RNAs in mice. In peripheral tissues, Igf2r sense and antisense RNAs show a reciprocal pattern of imprinting and DNA methylation between the two parental alleles: the sense RNA is monoallelically expressed only from the maternal promoter which is unmethylated in region 1, and the antisense RNA is derived solely from the paternal promoter which is unmethylated in region 2. The paternal promoter of sense Igf2r and the maternal promoter of antisense Igf2r are hypermethylated and are transcriptionally suppressed. In CNS, the genomic imprinting of Igf2r sense and antisense RNAs is uncoupled: both parental promoters of Igf2r gene coding for sense RNA are unmethylated and are biallelically used for transcription. In contrast, antisense RNA of Igf2r is derived only from the paternal allele that is unmethylated in region 2, while the methylated maternal allele is silent. Uncoupling of genomic imprinting of Igf2r sense and antisense RNAs in CNS correlates with DNA methylation of the appropriate promoter region, thus favoring the model of DNA methylation over that of antisense as the chief regulator of Igf2r genomic imprinting.
A new guaianolide artselenin (1) and a new dimeric guaianolide artselenoid (2), along with 10 known compounds, were isolated from the aerial parts of Artemisia selengensis. Their structures were elucidated by spectroscopic methods. Two-dimensional NMR techniques were used to make complete assignments for the 1H- and 13C-NMR chemical shifts of the two new guaianolides.
During the early developmental period of central nerves system, glucocorticoid can influence the activity of hypothalamic-pituitary-adrenocortical axis via glucocorticoid receptors, and modulate the process of neural survival, outgrowth and programming death process; In adult, glucocorticoid can regulate the expression of the factors which correlate with the plasticity of neurons; In aged individual, glucocorticoid can be harmful to nerves under most conditions. Therefore, glucocorticoid plays an important role in the development of neural system.
The human IGF2 and H19 genes are imprinted in most normal tissues. Alterations of genomic imprinting or loss of imprinting (LOI) have been observed in a number of malignant tumors. Although LOI has been linked to tumorigenesis, loss of IGF2 imprinting has also been observed in choroid plexus and leptomeninges in normal mouse brain. We have therefore analyzed the allelic expression of both IGF2 and H19 in human fetal brain and in different regions of human adult brain. In the brains of fetuses of 6-12 weeks gestation, both IGF2 and H19 were transcribed from both parental alleles. In contrast, strictly monoallelic expression of both IGF2 and H19 was observed in all other fetal tissues, suggesting a tissue-specific LOI in the central nervous system. In adult brain, LOI of IGF2 was region-specific. IGF2 was expressed from both parental alleles in the pons, but not in globus palludus, Raphe nucleus and hypothalamus. H19 expression was drastically reduced in adult brain compared to fetal brain, and was detectable only in the pons and globus palludus. In contrast to IGF2, the expression of H19 in adult pons was monoallelic. Examination of IGF2 promoter usage indicated predominant utilization of promoter P3 in all fetal and adult brain tissues. The LOI of IGF2 therefore reflects biallelic expression from the predominant promoter. IGF2 transcripts derived from the less abundant promoter P1, however, showed monoallelic expression in the adult pons. Our results suggest that IGF2 and H19 undergo ontogenetic changes in allelic expression and that there is dissociation of IGF2 and H19 imprinting in both fetal and adult human brain.
Reversible histone acetylation plays a central role in X chromosome inactivation. Histones are hypoacetylated on the heavily methylated inactive X chromosome and are hyperacetylated in the unmethylated "CpG islands" in animal genomes. We have investigated whether histone acetylation is involved in the regulation of the allelic expression of insulin-like growth factor II (IGF2), a maternally imprinted gene. HSK09, a human fibroblast cell line, retained normal monoallelic expression of IGF2 in culture. When these cells were treated with histone deacetylase inhibitors, sodium butyrate or trichostatin A, biallelic expression of IGF2 was observed from all of the promoters that are expressed. These results suggest that, in addition to DNA methylation, differential histone acetylation of two parental alleles may be another potential mechanism by which the imprinting of IGF2 is regulated, probably through changes in the local chromatin structure of the imprinted locus on chromosome 11p15.
The correlation between telomerase activity, telomere lengths, and cellular replicative capacity has led to the theory that maintenance of telomere lengths by telomerase acts as a molecular clock to control replicative capacity and senescence. Regulation of this molecular clock may have applications in the treatment of cell aging and tumorigenesis, although little is presently known about the regulation of telomerase activity. To investigate possible mechanisms of regulation, we examined telomerase activity and the expression of the human telomerase RNA subunit and the human telomerase reverse transcriptase protein (hTERT) during human fetal heart, liver, and kidney development. The human telomerase RNA subunit is expressed in all three tissues at all gestational ages examined. hTERT expression correlates with telomerase activity in the liver, where both are expressed at all ages surveyed, and in the heart, where both are present until the 11th gestational week but not thereafter. However, although telomerase activity in the kidney is suppressed after the 15th gestational week, the hTERT transcript can be detected until at least the 21st week. Reverse transcription-PCR using primers within the reverse transcriptase domain of hTERT show the presence of multiple alternately spliced transcripts in these tissues, corresponding to full-length message as well as spliced messages with critical reverse transcriptase motifs deleted. Of note, telomerase activity in the kidney is only present at those gestational ages when full-length hTERT message is expressed (until approximately week 15), with spliced transcripts continuing to be expressed at later stages of development. The tissue-specific and gestational-age dependent expression of hTERT mRNA seen in human development suggests the presence of at least two regulatory mechanisms controlling the activity of telomerase: transcriptional control of the hTERT gene and alternate splicing of hTERT transcripts.